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. 2006 Dec 29;351(4):1060-5.
doi: 10.1016/j.bbrc.2006.10.169. Epub 2006 Nov 7.

MNB/DYRK1A phosphorylation regulates the interactions of synaptojanin 1 with endocytic accessory proteins

Tatyana Adayev  1 , Mo-Chou Chen-Hwang, Noriko Murakami, Rong Wang, Yu-Wen Hwang
Affiliations

MNB/DYRK1A phosphorylation regulates the interactions of synaptojanin 1 with endocytic accessory proteins

Tatyana Adayev et al. Biochem Biophys Res Commun. .

Abstract

MNB/DYRK1A is a proline-directed serine/threonine kinase implicated in Down syndrome (DS). In an earlier screening, two proteins from adult rat brain, one 100kDa and the other 140 kDa, were found to be prominently phosphorylated by the kinase. The 100-kDa protein was previously characterized as an isoform of dynamin 1. In this study, we identified the 140-kDa protein as synaptojanin 1 (SJ1). MNB/DYRK1A phosphorylates SJ1 at multiple sites and produces complex behaviors in binding to amphiphysin 1 and intersectin 1 (ITSN1). However, the phosphorylation has little effect on the phosphatidylinositol phosphatase activity of SJ1. These results suggest that MNB/DYRK1A is involved in regulating the recruitment activity but not the phosphatase activity of SJ1. Our findings may be especially important in the etiology of DS because MNB/DYRK1A, SJ1, and ITSN1 are all located at or near the region of human chromosome 21, which is postulated to be involved in the disease.

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Figures

Figure 1
Figure 1
Phosphorylation of affinity-purified SJ1 and DY1 by MNB/DYRK1A. SJ1 and DY1 were purified from rat brain extract by the AMPH1(SH3) affinity chromatography as described in Materials and Methods. The AMPH1(SH3) domain binds primarily a 100-kDa and a 140-kDa proteins as revealed by Coomassie blue staining (A). The eluted sample was then analyzed for (B) DY1 (using antibody Hudy-1) and (C) SJ1 (using the anti-SJ1 antibody provided by Dr. P. S. McPherson) by immunoblotting. The same samples were also subjected to MNB/DYRK1A solid-phase assay (D). BE, crude brain extract; E, column elution; Std, protein standards.
Figure 2
Figure 2
Phosphorylation of native SJ1 by MNB/DYRK1A. Affinity-purified SJ1 (1.5 μg) was phosphorylated with the indicated amounts of MNB/DYRK1A under the native conditions as described in Materials and Methods. Stoichiometry of phosphate incorporation was calculated as the number of moles of phosphate per mole of SJ1. Each point represents the average of three independent phosphorylation assays.
Figure 3
Figure 3
The effects of MNB/DYRK1A phosphorylation on the binding of SJ1 to AMPH1 and ITSN1. Affinity-purified SJ1 (1.5 μg) was first phosphorylated with the indicated amounts of MNB/DYRK1A under the native conditions and analyzed for binding to (A) AMPH1(SH3) and (B) ITSN1(SH3) domains by the pull-down assay. The data were normalized to the unphosphorylated SJ1 before plotting and were the average of three independent trials. A representative of each binding assay (insert) was shown.
Figure 4
Figure 4
The effects of MNB/DYRK1A phosphorylation on the PI5Pase activity of SJ1. Affinity-purified SJ1 (1.5 μg) was first phosphorylated with the indicated amounts of MNB/DYRK1A under the native conditions. One-tenth of the phosphorylated SJ1 was then subjected to PI5Pase assay by using PI(4,5)P2 as the substrate. The PI5Pase activity was calculated as moles of phosphate released per mole of SJ1 per min.
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